We plan to continue our fractionation of tumor homogenates on DEAE-Sephacel in order to separate isozymes of both alkaline phosphatase and cyclic nucleotide phosphodiesterase. For alkaline phosphatase we will determine the inhibitions profiles of the separated activity peaks. In addition we will begin polyacrylamide gel electrophoretic analysis of these activity peaks using untreated fractions and fractions treated with various glycosidases; e.g. neuraminidase. Further, we will use donated antiserum to determine whether any cross-reactivity exists between the isolated activity containing peaks and the isozymes of liver, placental, and intestinal origins. Variations of the radiometric assay for phosphodiesterase suitable to the assay of microgram quantities of tissue have been evaluated. The most sensitive of these methods involved the assay of 5 microliters of sample volumes containing 1-100 micrograms of tissue extract. Tumor enzyme was found to be stable frozen at minus 76 degrees C or refrigerated overnight at a pH range of 4.8 to 9.0. Results of assays on lyophilized pieces, homogenates, and the supernatants and pellets of 27,000 x g centrifugations indicate that greater than 60% of the tumor enzyme is soluble. Kinetic studies demonstrate at least two forms of the enzyme are present in both malignant and benign samples, each with KMs in the 5 micro molar and 50 micro molar cAMP range. Preliminary fractionation data reveal two to four peaks of activity on DEAE-Sephacel, again with no significant difference appearing between the two tumor types.